Millions of people suffer from irreversible hearing loss due to hair cell degeneration as a result of aging and early exposure to hazardous environment such as noise. Unlike other species such as birds, the mammalian inner ear does not replace the damaged hair cells spontaneously. Any intervention aiming at replacing damaged hair cells requires understanding the developmental controls of the hair cells, so that by manipulating the determining factors it will be possible to induce the regeneration of hair cells. The development of the hair cell is controlled by proliferation of progenitor cells, initiation of cell fate determination and the terminal differentiation. Little is known, however, about the identity of genes and associated pathways that control the cell cycle exit of progenitor cells and the maintenance of the quiescent post mitotic status of the hair cells and the supporting cells. In this application we propose to accomplish fours aims. First, to test our hypothesis that the retinoblastoma gene (Rb) is critically involved in the cell cycle exit of the sensory epithelium in the inner ear. This will be accomplished using the conditional Rb knockout mouse model in which Rb is abolished in the sensory epithelial cells. Furthermore two hair cell- specific Rb conditional mouse models will be studied, to understand whether Rb is required for the maintenance of post mitotic hair cells. These mouse models will also provide us with the answers to questions including whether Rb is involved in the differentiation of hair cells, or whether functional hair cells can be produced in the absence of pRb function. Second, to test our hypothesis that Rb is required for the quiescent status of mature hair cells. The proliferation potential of mature hair cells will be studied, after acute deletion of Rb in the mature lox-P Rb utricle organ culture system. The newly derived hair cells will be studied to determine if they are functional, by dye FM1-43 uptake and patch clamping. The re-entry into the cell cycle by the mature hair cells to produce the functional hair cells will have enormous potential for hair cell regeneration for future therapy. Third, using the functional genomic approach, we will identify the pathways controlled by Rb in the inner ear. Identification of critical molecules involved in the Rb pathway will offer us the possibility to fine-tune the process controlling the hair cell post mitotic status. Lastly, by taking advantage of proliferating properties of differentiated Rb-null hair cells, we will establish cell lines enriched with hair cell properties. The cell lines will be characterized for hair-cell-specific gene expression. Such cell lines will be of great value to hearing research in general. Completion of the project will provide better understanding of the mechanism involved in cell cycle control of hair cells, and help to open new avenues in hair cell regeneration.